AU2015304820B2 - Novel bacterium of bacillus genus and uses thereof - Google Patents

Abstract

The present work relates to a novel microbe belonging to

Description

BACKGROUND OF THE INVENTION

The Earth's atmosphere is known to team with airborne microorganisms, though the high light intensities, extreme temperature variations, low concentrations of organic matter and scarcity of water, make the environment unsuitable for microbial growth. Biological material may contribute about 204),,22% and 10% to the total airborne particulate matter fey volume in remote continental, populated continental and remote maritime environments, respectively, Most of them originate from natural sources such as soil, lakes, animals and humans. Moreover, agricultural practices, health care units and industrial operations such as sewage treatment, animal rearing, fermentation processes, and food processing plants also emit viable microorganisms· into the environihe.nL

Bacteria form a large domain of single-celled, prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from cocci to rods and spirals. Bacteria are ubiquitous on Earth, growing in soil, acidic hot springs, radioactive waste, water, and deep in the Earth’s crust, as well as in organic matter and the live bodies of plants and animals. The bacilli are rod-shaped, gram-positive, sporulating, aerobic or facultative anaerobic bacteria. Most bacilli are saprophytes. Each bacterium creates only one spore, which, is resistant to heat, cold, radiation, desiccation, and disinfectants, The bacilli exhibit an array of physiological abilities that allow them to live in a wide range of habitats, including manyextreme habitats such as the desert sands, hot springs, and Arctic soils. species can be thermophilic, psychrophilie, acidophilus» alkaiiphilic, halotolerani, or halophilic and are capable of growing at various pH values, temperatures, and salt eoneentrations.

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Production of antimicrobial agents seems to be a general phenomenon for most bacteria. These bacteria produce an admirable array of microbial defence systems, including broadspectrum classical antibiotics, metabolic by-products such as organic acids, and lytic agents such as lysozyme. In addition, several types of protein exotoxins, and bacteriocins, which are biologically active peptide moieties with bactericidal mode of action, are also produced. The biological arsenal from microbes is remarkable in its diversity and natural abundance.

The search for new antimicrobial agents is a field of utmost importance. The development of resistance to antimicrobial agents is increasing at an alarming rate. Current solutions involve development of a more rational approach to antibiotic use and discovery of new antimicrobials.

Highly relevant patents

1. Novel bacterial strains and methods of controlling fungal pathogens (WO/2000/015761).

EMBODIMENTS OF THE INVENTION

Embodiments of the present invention provide a novel bacterium exhibiting antimicrobial and/or antifungal and plant growth promotion activity.

Other embodiments of the present invention provide for the isolation and identification of an extract of the novel bacterium, wherein the extract displays antimicrobial and/or antifungal plant growth promotion activity.

A further embodiment of the present invention provides an antimicrobial and/or antifungal and plant growth promotion composition or agent wherein the composition or the agent comprises the novel bacterium and/or the extract of the novel bacterium.

Other embodiments of the present invention provide a method of inhibiting the growth of pathogenic microbes and/or fungi by contacting the pathogenic microbes and/or fungi with an effective amount of the novel bacterium and/or an antimicrobial and/or antifungal and plant growth promotion composition and/or agent wherein the composition or the agent

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2015304820 25 May 2018

Yet another embodiment of the present invention provides use of the novel bacterium, an antimicrobial and/or antifungal composition or agent wherein the composition or the agent comprises the novel bacterium and/or the extract of the novel bacterium and/or a mixture of the novel bacterium and its extract, for inhibiting the growth of pathogenic microbes and/or fungi.

SUMMARY OF THE INVENTION

In a first aspect the present invention provides a novel bacterium belonging to a Bacillus species exhibiting antimicrobial and/or antifungal activity and plant growth promoting activity, wherein the novel bacterium is isolated Bacillus subtilis ssp. shriramensis having the accession number (MTCC-5674).

In a second aspect the present invention provides an extract of the novel bacterium belonging to a Bacillus species according to the first aspect, exhibiting antimicrobial and/or antifungal activity.

In a third aspect the present invention provides a pure culture of the novel bacterium according to the first aspect.

In a fourth aspect the present invention provides a process for producing the extract according to the second aspect, wherein the process comprises:

a. growing the Bacillus subtilis ssp. shriramensis having the accession number MTCC-5674 in a T3 medium having pH 6.8 in a shaking incubator at 30°C for 60 h; and

b. recovering the extract having antimicrobial and/or antifungal activity.

In a fifth aspect the present invention provides a composition comprising the novel bacterium according to the first aspect, wherein the composition has antimicrobial and/or antifungal activity and plant growth promoting activity at a concentration between about

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5x10⁵ cfu/ml to about 5xl0⁷ cfu/ml of the bacterium. In a sixth aspect the present invetion provides a composition comprising the extract according to the second aspect, wherein the composition has antimicrobial and/or antifungal activity at a concentration of between about 4 pg/μΙ to about 20 pg/μΙ of the extract.

In a seventh aspect the present invention provides a composition comprising the novel bacterium according to the first aspect and the extract according to the seocnd aspect, wherein the composition has antimicrobial and/or antifungal activity and plant growth promoting activity.

In an eighth aspect the present invention provides a method for inhibiting growth of pathogenic fungi and/or bacteria, wherein said method comprises contacting the pathogenic fungi and/or bacteria with an effective amount of between about 5xl0⁵ cfu/ml to about 5xl0⁷ cfu/ml of the novel bacterium according to the first aspect or a composition according to the seventh aspect.

In a ninth aspect the present invention provides the novel bacterium according to the first aspect or the extract according to the second aspect when used for the preparation of a composition for inhibiting the growth of pathogenic fungi and/or bacteria.

An aspect of the present invention is to provide an isolated, novel bacterium which is useful in producing antimicrobial and/or antifungal metabolites or agents.

One aspect of the present invention is to provide a novel form of bacterium belonging to Bacillus species which is designated as Bacillus subtilis ssp. shriramensis having accession number (MTCC-5674). In particular, the novel bacterium disclosed in the present work is capable of exhibiting distinct antimicrobial and/or antifungal and plant growth promotion property.

Another aspect of the present invention is to provide a process for the production of an antimicrobial and/or antifungal and plant growth promotion composition or agent wherein the composition or the agent comprises Bacillus subtilis ssp. shriramensis (MTCC-5674) and/or the extract of the Bacillus subtilis ssp. shriramensis (MTCC-5674).

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There is provided a composition comprising Bacillus subtilis ssp. shriramensis (MTCC5674). The composition may further comprise pharmaceutically acceptable excipients, diluents and/or carriers.

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There is provided a composition containing an extract of Bacillus subtilis ssp. shriramensis (MTCC-5674). There is also provided a composition comprising an aqueous extract of Bacillus subtilis ssp. shriramensis (MTCC-5674). The composition may further comprise pharmaceutically acceptable excipients, diluents and/or carriers.

There is provided a method for inhibiting the growth of pathogenic microbes and/or fungi by contacting the pathogenic microbes and/or fungi with an effective amount of Bacillus subtilis ssp. shriramensis (MTCC-5674) or the extract of Bacillus subtilis ssp. shriramensis (MTCC-5674). The Bacillus subtilis ssp. shriramensis (MTCC-5674) and/or the extract of Bacillus subtilis ssp. shriramensis (MTCC-5674) may optionally contain one or more additional antimicrobial and/or antifungal and plant growth promoting agents.

There is provided in the present invention the use of Bacillus subtilis ssp. shriramensis (MTCC-5674) and/or the extract of the Bacillus subtilis ssp. shriramensis (MTCC-5674) in the formulation of an antimicrobial and/or antifungal and plant growth promotion composition or agent for inhibiting the growth of pathogenic microbes and/or fungi.

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DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel microbe belonging io PaciPtis family designated as

Pacd<'>

«Λ having an accession number (MTCC-5674) and a method of producing an antimicrobial and/or antifungal and plant growth promotion composition or agent wherein the composition or the agent comprises Bac/Bus subtilis ssp, (MTCC-5674) and/or the extract of the subtilis ssp. skrirwtiensis (MTCC-5674).

The present invention also provides a method of inhibiting the pathogenic microbes and/or fungi by contacting the microbes and/or fungi with an effective amount of the novel bacterium, Pacilfus subtilis ssp. .shrerawnriv (MTCC-5674) and/or the composition comprising the novel bacterium: or its extract.

The present invention also provides use of Bacillus .v«b/i7R ssp. sfoinwiensis (MTCC-5674), and/or an antimicrobial and/or antifungal and plant growth promotion composition or agent, comprising the novel bacterium Bacillus ten?////,r ssp. shrinimeasis (MTCC-5674) and/or the extract of the Bacillus snbB/A ssp. s/vvTamem'A (MTCC-5674) for inhibiting the pathogenic microbes and/or Ringb

The novel mubB/N ssp. (MTCC-5674) may be utilized for the mass production of antimicrobial and/or antifungal and plant growth promotion eomposition/preparation/agent by culturing Bacillus subtilis ssp, ^ArZrayienwT in the suitable growth medium under favourable conditions.

Through deep and careful researches, the inventors have surprisingly found, isolated and cultured a novel bacterium, which can produce a novel agent. Through detailed experimental researches, the inventors have also invented a method of producing the said novel agent from tbe said novel microorganism.

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Brief Description of Accompanying Drawings

Fig. I - Plate showing isolation and purification of subtilis ssp. s/raw/aw (MTCC5674) (A) Mother culture plate showing bacterial growth along with fungal mycelium: (B)

Purification of wfeti/A ssp. shriramznsis (MTCC-5674) from bacterial eolony(s) in (A).

Arrow indicates putative bacterial colony.

Fig, 2 - Clone of one of the purified colonics of Bacillus subfilis ssp, (MTCC5674) showing inhibition of .growth of Ram/ww exy,fp&rwn mycelium.

Fig. 3 - Microscopic picture of vegetative bacterial cells of' Bneti/us subtilis ssp. shrirmnemfev (MTCC-5674) along with spores.

Fig, 4 - Plate showing actively growing: colonies of BdeB&s sttbtilis ssp, .-fMimJwa (MTCC5674).

Fig. 5 - Rod shaped &?i.7/.ti<s iubiilli' ssp. shriniwensis (MTCC-5674) under light microscope. Fig, 6 - Picture showing results of catalase test: (A) Negative control; (B) Positive control and (C) Bacillus .iublitb? ssp. shrint/newtis' (M7'CC-5674) showing positive results for catalase activity,

Fig. 7 - Plate showing amylolytic activity of Bacillus subiitls ssp, shrirauumsis (MTCC-5674) culture filtrate.

Fig. 8 » Picture showing results of O/F (Oxidation-Fermentation) test (A) Negative control; (B) Bacillus subtilis ssp. shrlramensis (MTCC-5674) showing color change only at the top portion of the medium; (C) Positive control.

Fig. 9 - Picture showing results of Hydrogen sulphide production test (A) Negative control; (3) Roud/us stdm'fe ssp, shrnwicnsri’ (MTCC-5674) and (C) Positive control Pig. Γ0 - Picture showing results of SDS-PACB of the concentrated- culture nitrate of 3nctih« <$·«&&/& ssp. (MTCC-5674),

Fig. 11 - Culture plates showing antimicrobial and/or antifungal activity displayed by (A) Bacdzi»· subii/is ssp. .s7/rira.’??e«,rD (MTCC-5674) colony and (Bi Bacillus .«mri/H ssp. sfirirnwws (MTCC-5674) culture filtrate, fterflfflt axysporum culture was used as test fungus.

Fig. 12 - Picture showing results of MIC assay of antimicrobial and/or antifungal compound by the tube dilution method. I to C-2: Pictures of /hmrrww axysparuw spores (observed under light microscope), after incubating in PD3 containing different concentrations of antimicrobial and/or-antifungal agent 1-28 (Dilutions HI to 1:.100), C-1 - Spores in antimicrobial and/or

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PCT/IN2015/000294 antifungal agent (Crude); C-2 - Control (Spores in PDB broth without antimicrobial and/or antifungal agent).

Fig. 13 - Picture showing results of MIC assay of antimicrobial and/or antifungal agent by agar diffusion method. 1 to C-2: Pictures of Fusarium oxysporum mycelium growing on test plates.

1-28 Dilutions of antimicrobial and/or antifungal agent at 1:1 to 1:100 (v/v), Cl - Well containing antimicrobial and/or antifungal agent (Crude); C2 - Control well containing PDB; C3 - Control well containing 70% saturated ammonium sulfate.

Fig. 14 - Picture showing effect of antimicrobial and/or antifungal agent on spores of Aspergillus niger. (A, B & C) Spores of Aspergillus niger showing normal germination in PDB media; (D) Spores of Aspergillus niger failed to germinate in PDB media containing Bacillus subtilis ssp. shriramensis (MTCC-5674) extract.

' Fig. 15 - Plate showing antimicrobial and/or antifungal activity of cell lysate against Fusarium oxysporum·, (1) Well containing only lysozyme (to check the effect of lysozyme on fungus Fusarium oxysporum ) and (2) Well containing cell lysate of Bacillus subtilis ssp. shriramensis (MTCC-5674).

Fig. 16 - Plate showing assay of antimicrobial and/or antifungal activity of Bacillus subtilis ssp. shriramensis (MTCC-5674) cells/extract against diverse types of plant pathogenic fungal and bacterial species. A. Fusarium oxysporum, B. Sarocladium oryzae C. Trichoderma viridae D. Colletotrichum capsicii E. Exerohilum turcicum F. Rhizoctonia solanii G. Macrophomina phaseolina H. Xanthomonas oryzae

Fig. 17 - Plate showing results of antimicrobial and/or antifungal activity of Bacillus subtilis ssp; shriramensis (MTCC-5674) extract on germination of rice seeds in presence of Fusarium oxysporum. (A) Rice seed treated with fungus Fusarium oxysporum spores; (B & C) Rice seeds treated with fungus Fusarium oxysporum and Bacillus subtilis ssp. shriramensis (MTCC-5674) extract.

Fig. 18 - Plate showing results of experiments to show absence of pathogenicity of Bacillus subtilis ssp. shriramensis (MTCC-5674) on various plant species. (A) Rice, (B) Cotton, (C) Tobacco, (D) Com, and (E) Tomato.

Fig. 19 - Picture showing results of experiments to show action of Bacillus subtilis ssp. shriramensis (MTCC-5674) as bio-control agent. (A) Tomato plant infected with Rhizoctonia solani (NFCCI-3194) fungus. (B) Tomato plant with Rhizoctonia solani (NFCCI-3194) and Bacillus subtilis ssp. shriramensis (MTCC-5674) and (C) Control tomato plant (without

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Rhizactatria solani fungus, and Bacillus subiilis ssp. shriramensis (MTCC-5674).

Fig. 20 - Plates showing (1) Penicillium oxalicum (NFCCI-1997) fungal colonies and (2)

Bacillus subiilis ssp, shriramensis (MTCC-5674) pure colonies,

Fig. 21 · Plate showing corn seeds coated with various formulations of antimicrobial/antifurjgal agent Bacillus subiilis ssp. shriramensis (MTCC-5674). 1. (Control-!) Seeds treated with the formulation which has no fungal pathogen and bio-control agent; 2, (Control-2) Seeds treated with the formulation which has no bio-control agent; 3. (Control-3) Seeds treated with the formulation which has commercial fungicide “Carbendazim WP50; 4a. Seeds treated with the formulation which has Bacillus subiilis ssp, shriramensis cells (5xl0⁴ cfu); 4b. Seeds treated with, the formulation which has Bacillus subiilis ssp. shriramensis ceils (5x10' cfu); 4c. Seeds treated with the formulation which has Bacillus subiilis ssp, shriramensis cells (5x10^e cfu); 4d, Seeds treated with the formulation which has Bacillus subiilis ssp. shriramensis cells (5xl0⁷ cfu) and 5. Seeds treated with the formulation which has only Bacillus subiilis ssp. shriramensis cells (5xl0⁷cfu).

Fig. 22 - Plate showing results of bio-control activity after 2 weeks of incubation, 1. (Control-1) Seeds treated with the formulation-1 which has no fungal pathogen and antifungal agent; 2. (Control-2) Seeds treated with, the formulation-2 which has no bio-control agent: 3. (Control-3) Seeds treated with the formulation-3 which has commercial fungicide 'Carbendazim WP59”; 4a, Seeds treated with the formnlation-4a which has Bacillus subiilis ssp, shriramensis (5x1 ()⁴ cfu); 4b. Seeds treated with the formulalion-4b which has Bacillus subiilis ssp. shriramensis cells (5xKP cfu): 4c. Seeds treated with the formulation-4c which has Bacillus subiilis ssp, shriramensis cells (5x1 0^& cfu) ; 4d. Seeds treated with the formulation-4d which has Bacillus subiilis ssp. shriramensis cells (5xl0⁷ cfu) and 5, Seeds treated with the formulation-5 which has only Bacillus subiilis ssp. shriramensis cells (5x10⁷ cfu).

Fig. 23 - Plate showing results of bio-control activity after 4 weeks of incubation, 1. (Control-1) Seeds treated with the formulation-1 which has no fungal pathogen and antifungal agent; 2, (Coiitrol-2) Seeds treated with the formulation-2 which has no bio-control agent; 3. (Control-3) Seeds treated with the formulation-3 which has commercial fungicide “Carbendazim WP50”; 4a. Seeds treated with the formulauon~4a which has Bacillus subiilis ssp, shriramensis (5x10* cfu); 4b. Seeds treated with the formu!aGon-4b which has Bacillus subiilis ssp. shriramensis cells (5x10* cfu); 4c. Seeds treated with the formuiation-ic which has Bacillus subiilis ssp, shriramensis cells (5x10^c cfu); 4d, Seeds treated with the formulation-id which has Bacillus •7

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PCT/IN2015/000294 subfitti ssp. cells (5x10^; cfu) and 5, Seeds treated with the formulation-? which has only ;/E?cf//«s ssp, .y&?7ra?zie.uffy cells (5xW⁷C'fo),

Fig,, 24 - Assay of antifungal and/or antimicrobial activity against a variety of human pathogenic fungal species, Α» B and C - Assay of antifungal and/or antimicrobial activity against PemciiiiiiM spp, (A) Penicifiwm spp. fungal colonies (B) Mycelium and (C) Effect of ZfocEfos' wz/b’B- ssp. s/iz-twzmm/s' (MTCC-5674) culture filtrate. D, E and F - Assay of antifungal and/or antimicrobial activity against zi.Ypcz'gZ/Z.’z.r/Ζζζν/ζτ (D) Fnngal colonies (E) Mycelium and (F) Effect ol Bocr/Zus .wm/EC ssp. .s'bz-zz7.?z??iniri.Y (MTCC-5674) culture filtrate, G, H and 1 - Assay of antifungal and/or antimicrobial activity against Aspergillus ?u'ger (G) Fungal colonies (Ti) Mycelium and. (D Effect of Bucifisis subti/is ssp. .YhzvMzzuezrffv (MTCC-5674) culture filtrate, ,fi K. and E - Assay of antifungal and/or antimicrobial activity against unknown fungus causing skin infection, (j) Fungal colonies (K) Conidia and (L) Effect of /InoZ/iUs· svi/m'/B ssp. s'A?'0’f.iz«ensA (MTCC-5674) culture filtrate.

Fig,25 - Effect of Iteeilhus .y/Bm'/A ssp. ^Az-Zz-ovxezwB (MTCC-5674) formulation on growth and development of Corn. The corn seed treated with formulation containing Baci/ii/s subtilis ssp, sbrisivnensis (MTCC-5674) showed higher growth rate, biomass and grain yield.

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ISOLATION AND IDENTIFICATION OF THE NOVEL· BACTERIUM

The inventors collected air samples from 18 different locations in Hyderabad and Patancheru (Telangana, India) while conducting a study on air flora. Disposable petri plates containing media (T3 Medium, Travers, ci aL· 1987) were prepared in the laboratory» and exposed to air at different locations. The exposed plates were sealed and incubated at 30° C in lab incubator. In one of the plates exposed to air in Patancheru. area, a bacterial colony surrounded by fungal mycelium was observed (Fig, 1A), Despite continued incubation, the clearance zone was maintained and growth of fungal mycelium remained restricted to the periphery of clearance zone. The microorganisms from this colony were subjected io purification by using standard methods ol microbiology (Fig, IB), The individual colonies were tested against a common fungus Eiswruw avjmwnwm (Fig. 2),

One of the colonies showed inhibition of fungal growth, and a clearance zone was observed ( Fig. 2). Microscopical examination of the bacteria from the .colony revealed a rod shaped motile bacterium (Fig, 5). After six days of incubation in the culture medium the bacteria produced spores. The colonies of the bacteria were mucoid, raised, circular, smooth, and creamy to offwhite in color (log. 4), and cells showed variable gram staining (Fig, 5).

A range of biochemical tests including carbohydrate fermentation, catalase activity, oxidationfermentation test, starch hydrolysis, hydrogen sulphide production test, oxidase activity test, desoxyehoiate agar test were carried out. The results of these tests confirmed that the bacteria is catalase positive, possess amylase activity, strongly aerobic, does not produce hydrogen sulfide, oxidase positive and gram variable, for identification of bacteria 16S DMA sequencing and FAME analysis was carried out. The results of both studies showed that the bacteria is showing 0.37% difference in 16S DNA sequence and FAME similarity index of 0.827; with Raci/fas subtiiis ssp. xubfi/ff and 0.$4% difference in I6S DNA. sequence and FAME similarity index of 0.749 with Jfacifh® atraphaeuy.'T.hus, the results suggest that this bacterium is related to .Oiirf/m.v tiitoitis and Bacfftus airophumo·, hut not identical to any of the catalogued bacterial species in ATCC collection.

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Results of 16$ DNA sequence comparison

Match	% Difference	Length	Library Entry Name
1	0,19	535	Bacillus subtilis ssp, .rnb/tZ/.v
2	0,84	535	Bacillus altopbaeus
3	1.03	535	Bacillus amylaliqui&cserts

Results of FAME analysis comparison

S, No,	Similarity Index	Library Entry Name J
1	0.827	Bacillus subtilis j
7 Λ·	0.749	Bacillus atropitaeus J

The isolated bacterium is a new member of sub-species of the genus BacHZifrsr. According to bacterial nomenclature convention, the novel bacterial species was named as Bacillus «ιά/ή’/ί ssp. sivuraw e/is A. The bacterium is deposited in the Microbial Type Culture Collection (MTCC) at 1MTECH, Chandigarh, India, The deposition number of this novel species is (MTCC-5674),

Characteristic Features of the Novel (MTCC-5674) provided by the present invention

The bacterium is a rod shaped measuring 2,45x0,88 pm, motile, spore forming, gram variable; colonies are smooth, mucoid, off-white to ereamish in early stages but turn wrinkled on prolonged, incubation, The bacterium transforms into spore as the nutrients in the medium having accessien/depQshion number deplete, normally the process of sporulation takes place in 4 days of incubation in 10 ml medium containing 100 pi of 5xl0⁸cells-inoculum in a 25x150 mm culture tube at 30 °C and shaking at

200 rpm,.

The novel bacterium, Bacillus subtilis ssp. sfoirameusis having accession number (MTCC5674) exhibits antimicrobial and/or antifungal activity, The extract of the novel bacterium, Bacillus: subtilis ssp, having accession number (MTCC-5674) exhibits antimicrobial and/or antifungal activity, The range of potential applications and uses of the bacterium are extensive.

'The present invention provides a method of producing the antimicrobial and/or antifungal extract from the novel bacterium. Bacillus subtilis ssp. s&wmnezwY having accession number (MTCC-5674).

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Production and Isolation »f Ajfihnicrobiai andM.Anfifan^aiAgcjrt .Composition of culture medium for the gro\vtL<.,gQgte.T»ferife..ssp· A/?rhWhg»yE..haying agcg.8sios.nomfc.er (M;TCC^5674LHag.foHows

L . Tryptone : 0,32% (w/v)

2. Tryptose : 0,24 % (w/v)

3. Yeast Extract : 0.18 % (w/v)

4. NaEbPOU-feO : 0.044 M

5. NaaHPCU : 0.062 M •6, MaCb : 0.000 5% (w/v) pH = 6.8 '

I. The medium was prepared as per the method given in Annexure -1 (I) and 100·ml aliquots were transferred into 500 ml conical flasks, The media was sterilised by autoclaving at 121°C for '15 min.

Each flask was inoculated with a single pure colony of Bacillus (MTCC-5674) and incubated at 30°C, 200 rpm for 60 hours.

Following the growth of bacteria in T3 broth tor 60 hours, the culture was centrifuged at 12()00 rpm for 10 min at 4 °C. The supernatant was collected and. filtered using 0,22 nm disc filter (Millipore/Sartorius). The filtrate was preserved under appropriate storage conditions for detailed experiments to study antimicrobial and/or antifungal activity.

The present invention particularly provides a novel microorganism, Bacillus xubfi/H ssp. s'hriramsasis having accession number (MTCC-5674) and a method for the production of antimicrobial and/or antifungal composition from the novel bacterium and/or its extract or a mixture of the novel, bacterium and/or its extract.

One embodiment of the present invention provides an. isolated novel bacterium belonging to //«c/i/ns suborns· ssp. exhibiting antimicrobial and/or antifungal activity, having accession number (MTCC-5674).

In one embodiment of the present invention is provided the novel bacterium designated as Eud/fos rfo/ms ssp. 5/ir««w having accession number (MTCC-5674), in another embodiment of the present invention there is provided a pure culture of the novel

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PCT/IN2015/000294 bacterium. Bacillus subtilis ssp, shriramcttsls having accession number (MTCC-5674).

in one embodiment of the present invention there is provided an extract of the novel bacterium,

Bacillus subtilis ssp. shrireitnensis basing accession number (M.TCC-5674) wherein the extract exhibits antimicrobial and/or antifungal activity.

In another embodiment of the present invention there is provided an extract of the novel bacterium, Bacillus subtilis ssp. 57irfoawe«57.v having accession number (MTCC’5674) wherein the extract exhibiting antimicrobial and/or antifungal activity is an aqueous extract. In yet another embodiment of the present invention there Is provided a process for the production of the extract of the'novel bacterium, .Soer/imr subri/ri ssp. .v/wvm morris having accession number (MTCC-5674.) where in the process comprises growing the novel bacterium, Βοο/Ρνν subtilis ssp, shriramensis having accession number (MTCC-5674) in a nutrient medium and recovering the extract having antifungal activity by using conventional methods.

In another embodiment of the present invention there is provided a process for the production of the extract of the novel bacterium. rio£u7/m' xabri/h ssp, s/mmowtw»· having accession number (MTCC’5674) wherein the process comprises growing the novel bacterium, /hreri/nx ΛΙ/w ssp, s/irireimiMsis havina accession number (MTCC-5674) under aerobic conditions.

‘ * u.

in yet another embodiment of the present invention there is provided a process for the production of the extract of the novel bacterium, .«fori/rs ssp. sbri.nmnen.sri having accession number (MTCC-5674) wherein the process comprises growing die novel bacterium, .&.7θήϊίί,γ «ibri/ί,ν ssp, shrireimensht in a nutrient medium, recovering the extract having antimicrobial and/or antifungal activity and optionally comprises concentrating the extract using conventional -methods.

In one embodiment of the present invention, there is provided, a composition comprising the novel bacterium, Bacittos subtilis ssp. iMres»/® having accession number (MTCC’5674) wherein the composition has antimicrobial and/or antifungal activity.

in another embodiment of the present invention, there is provided a composition, comprising the extract of the novel bacterium, Bacillus xubo’/ri- ssp. sht'iramettsls having accession number (MTCC’5674) wherein the composition has antimicrobial and/or antifungal activity, in another embodiment of the present invention, there is provided a composition comprising the

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PCT/IN2015/000294 novel baeterium_s Bacillus subtilis ssp, shriramensis having accession number (MTCC-5674) and the extract of the novel bacterium. Bacillus subtilis ssp, shriramensis having accession number (MTCC-5674) wherein the composition has antimicrobial and/or antifungal activity.

In one embodiment of the present invention. there is provided a composition comprising the novel bacterium. Bacillus subtilis ssp, shriramensis having accession number (MTCC-5674) anchor an extract of Ute said novel bacterium, Bacillus subtilis ssp, shriramensis, or a combination thereof that optionally comprises one or more antimicrobial and/or antifungal agents. '

In another embodiment of the present invention, there is provided a composition comprising an extract of the novel bacterium, Bacillus subtilis ssp. shriramensis having accession number (MTCC-5674) that optionally comprises one Or more antimicrobial and/or antifungal agents.

in yet another embodiment of the present invention there is provided a composition comprising the combination of the novel bacterium. Bacillus subtilis ssp. shriramensis having accession number (M TCC-5674) and its extract that optionally -comprises one or more antimicrobial and/or antifungal agents.

In one embodiment of the present invention there is provided a composition comprising the novel bacterium, Bacillus subtilis ssp. shriramensis having accession number (MTCC-5674) or an extract of the novel bacterium, Bacillus subtilis ssp, shriramensis having accession number ’CC-5674) or a combination thereof that optionally comprises agriculturally or laceutically acceptable carrier. .

In another embodiment of the present invention there is provided a composition comprising the novel bacterium, Bacillus subtilis ssp. shriramensis having accession number (MTCC-5674) that optionally comprises agriculturally or pharmaceutically acceptable carrier. .

In yet another embodiment, of the present invention there is provided a composition containing an extract of the novel bacterium. Bacillus subtilis ssp. shriramensis having accession number (MTCC-5674) that optionally comprises agriculturally or pharmaceutically acceptable carrier.

In still another embodiment of the present invention there is provided a composition comprising, the combination of the novel bacterium, Bacillus subtilis ssp. shriramensis having accession

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PCT/IN2015/000294 number (MTCC-5674) and an extract of the said .novel bacterium, BacB/us subtilis ssp. shriramensis, which optionally comprises agriculturally acceptable carrier (See Annexure III), in one embodiment ofthe present invention there is provided a method for inhibiting growth of pathogenic fungi and/or bacteria, wherein said method comprises contacting the pathogenic fungi and/or bacteria with an effective amount of the novel bacterium, Bacillus subtilis ssp. shriramensis having accession number (MTCC-5674) or a composition comprising the said novel bacterium, or its extract or a combination thereof.

In one embodiment of the present invention there is provided a method for inhibiting growth of pathogenic fungi and/or bacteria, wherein said method comprises contacting the pathogenic fungi and/or bacteria with an effective amount of the novel bacterium, Bacillus subtilis ssp, shriramensis having accession number (MTCC-5674),

In another embodiment of the present invention there is provided a method tor inhibiting growth of pathogenic fungi and/or bacteria, wherein said method comprises contacting the pathogenic fungi and/or bacteria with an effective amount of a composition comprising the novel bacterium, Bacillus subtilis ssp, .r/irmnmmyfv having accession number (MTCC-5674).

In yet another embodiment of the present invention there is provided a method for inhibiting growth of pathogenic fungi and/or bacteria, wherein said method comprises contacting the pathogenic fungi and/or bacteria with an effective amount of a composition comprising, an extract of the novel bacterium, Brnc/rio subB/ri ssp. shriramensis (MTCC-5674) wherein the extract has antimicrobial and/or antifungal activity.

In yet another embodiment of the present invention there is provided a method for inhibiting growth, of pathogenic fungi and/or bacteria, wherein said method comprises contacting the pathogenic fungi and/or bacteria with an effective amount of a composition comprising the novel bacterium, Bacillus subtilis ssp. (MTCC-5674) and an extract of the said novel bacterium, Bacillus .mbri/ri ssp. xb/a>£?/«cw/.Y, wherein the extract has antimicrobial and/or antifungal activity.

In one embodiment of the present invention there is provided a use of the novel bacterium, Bacillus subtilis ssp. shriramensis having accession number (MTCC-5674) or a composition comprising the said novel bacterium or its extract or a combination thereof, for Ute preparation

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PCT/IN2015/000294 of an antimicrobial and/or antifungal composition for inhibiting the growth of pathogenic fungi:

anchor bacteria.

In another embodiment of the present invention there is provided a use of the novel bacterium, Bacillus suhtilis ssp, shriramensis having accession number (MTCC-5674) for the preparation of an antimicrobial and/or antifungal composition for inhibiting the growth of pathogenic fungi and/or bacteria.

a use of the composition sis (MTCC-5674) for the inhibiting the growth of

In another embodiment of the present invention there is provided comprising the novel bacterium, Bacillus subtflis ssp, shrtramen preparation of an antiinicrobial and/or antifungal composition for pathogenic fungi and/or bacteria.

In another embodiment of the present invention there is provided a use of the composition comprising an extract of the novel bacterium. Bacillus subiilis ssp. xhriramensis (MTCC-5674) for the preparation of an antimicrobial and/or antifungal composition for inhibiting the growth of pathogenic fungi and/or bacteria.

in another embodiment of the present invention there is provided a use of the composition comprising the extract of the novel bacterium, Bacillus subiilis ssp, shriramensis (MTCC-5674) and an extract of the said novel bacterium, Bacillus sub fills ssp, stimainetisis, for the preparation of an antimicrobial and/or antifungal composition for inhibiting the growth of pathogenic fungi and/or bacteria, ..

In another' embodiment, there is provided a pharmaceutical and agriculturally effective composition comprising the novel bacterium. Bacillus subiilis ssp. shrira/tK’tisis having accession number (MTCC-5674).

In another embodiment, there is provided δ pharmaceutical and agriculturally effective composition comprising the extract of the novel bacterium, Bacillus subiilis ssp. shriratriwsis (MTCC-5674), in yet another embodiment of the present invention there is provided a method of producing the said effective composition from the novel bacterium, Bacillus subtilix ssp, shrlraTnetisis having accession number (MTCC-5674).

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In. yet another embodiment of .the present invention, the steps and time required for the production of the said composition/extract are kept at tire minimum duration coupled with the maximum recovery of the compound. - .

Other advantages or benefits of the present invention

The bacterium Bacillus subtilis ssp, shriramensis (MTCC-5674) along with antimicrobial and/or antifungal agent also produces strong thermophilic protease and amylase which are active even after exposure to high temperature i.e., 121 °C for ISmin.

The present invention is further explained by the following examples. However, the present invention Is not limited to these examples in any manner. The following examples is intended to illustrate the working of disclosure and not intended to take restrictively to apply any limitations on the scope of the present invention. Those persons skilled in the art will understand that the equivalent substitutes to the specific substances described herein, or the corresponding improvements are considered to he within the scope of the invention.

Detailed Methodology Is explained in the following examples;

The methods employed in the present work are well-known in microbiology with the respective parameters varied and optimized for the present study.

Example 1

-1 Collection and preliminary screening of air samples

Air sampling was carried out at different locations in Hyderabad and Patancheru (Telangana, India), Disposable Petri plates containing T3 medium were prepared in the laboratory and exposed to air at different locations. The exposed plates were sealed and incubated at 30°C in lab incubator, in one of the plates exposed to air in Patanelieru area, a bacterial colony surrounded by fungal mycelium was observed.

1.2 Preliminary screening of air samples for antimicrobial and /or antifungal activity

Despite continued incubation the clearance zone was maintained and growth of fungal mycelium remained restricted to the periphery of clearance zone. 1'he microorganisms mom this colony were subjected to purification by using standard methods of microbiology (Pig. IB). The individual colonies were tested against a common fungus Fusarium oxysporum (Pig. 2). One of the colonies showed inhibition of fungal growth and a clearance zone was observed (Fig. 2), '

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-3 Screening„of noyeUsoiate

Evaluation of the bacteria under microscope revealed that it is a rod shaped motile bacterium (Fig, 5). After six days of incubation the bacteria produced spores.

The colonies of the bacteria were mucoid, raised, circular, smooth, and creamish to offwhite in color (Fig, 4) and the cells showed variable gram staining,

Example 2

-1 Chamcierlzaf mg arid Jdgnti&ato

2.1.1 Charnel erizationi^hejmyeljsqla^^ §ccessiooymberfMTOC^6^

A range of biochemical tests, including carbohydrate fermentation, catalase test, oxiddtionfermentation test, starch hydrolysis, hydrogen sulfide production test. mtidase activity test, were carried out, The results of these tests confirmed that the bacterium is catalase positive, arnyiase positive, oxydase positive and strongly aerobic,

2.M.1

Colonies of Bacillus subtilis ssp, shriramensis (MTCC-5674) are mucoid, raised., circular, smooth, and creamish to off-white in color (Fig, 4).

2.F1.2 Sdtuig^tecteristo

Bacillus subtilis ssp, .mrftumemA (MTCC-5674) shows optimum growth at 30X1 (can grow from 15 C to 55C). As it is an aerobic bacterium, it requires adequate oxygen lor its growth, needs continuous shaking lor culturing in broth.

i 2,1,1.3 .Celljnprphplogy

Bacillus subtilis ssp. shriramensis (MTCC-5674) ceils are rod shaped, dipiobacilii and motile (Fig, 5).

2,1.1.3.1 Contpafspri.M. colony...growth, and juorphoipgy^^./Mgftfe..rgdq/ff.„ssm

	ft. sunft/A ssp, .sfenrmmms'A	B. subtilis ssp, Stf'OO/fe	ft nn'pij/mem
Colony Morphology	Mucoid, circular, smooth, (rough after prolonged incubation), 2,()-44) mm in diameter.	Mucoidxireular, entire, opaque, 2.0-4.0 mm in diameter	Opaque, smooth, circular, entire and 1.0-2.0 mm diameter (Nakamura L.K. · 1989),

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Colony Color

Cream to off-white

Off-white to brown

Dark brown to black

2.1.1.4 Catalase test Material • Culture tubes of Bacillus subtilis ssp. shriramensis (MTGC-5674) • Hydrogen Peroxide Method • Three tubes containing LB medium were labelled as “test”, “positive control” and “negative control” a loop full of Bacillus subtilis ssp. shriramensis (MTCC-5674), Escherichia coli and Streptococcus pneumonia were inoculated in the tubes respectively. Following incubation at 30°C for 24 hours, few drops of hydrogen peroxide were added in all the tubes and observed for formation of bubbles.

Result

Gas bubbles were formed both in “test” and “positive control” tubes indicating that the Bacillus subtilis ssp. shriramensis (MTCC-5674) is catalase positive (Fig. 6).

2.1.1.5 Starch Hydrolysis Material • Bacillus subtilis ssp. shriramensis (MTCC-5674) culture filtrate • Starch agar plates • Iodine • Incubator Method

The starch agar medium was prepared as per the method provided in the Annexure - I (VI). Two wells were made at equal distances in the plate containing starch agar medium and labelled as “test” and “negative control”. An aliquot of 500 μΐ each of Bacillus subtilis ssp. shriramensis (MTCC5674) culture filtrate and sterile distilled water were dispensed into the wells labelled “test” and “negative control”. The plate was incubated at 50°C for 4 hours.

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BsSilU

After 4 hours of incubation, the blue color surrounding the test well disappeared indicating that the .Bacillus suljfliis ssp- (MTGC5674) culture filtrate has amylolytie activity. No change in the blue color was observed in the area surrounding control well (Fig. 7),

2.1.1.6 OdLiO&hbfi^^

Material » Hugsh Leifson’s OF Basal Medium * Test tubes « £.eon culture « Zte/Zte .subd'/ft' ssp, s/Wnwmiy (MTCC-5674) ® incubator

Method

Three tubes containing Hugsh Leifson’s OF basal medium (OFBM) (Annexure - I (VH)) were labelled as “negative control”, “positive control” and “test” and a loop .full of .,4fcu//ge«m? faecatis, Escherichia coti and Bntu&y subfi/ft' ssp, iArtowattf (MTCC-5674) was inoculated in the tubes rcspecti vely, The tubes were Incubated at 30^cC for 48 hours and observed for change of color.

Besftlt ft has been concluded from the observations that the test organism (Bacillus subtilis ssp. shriramensis (MTCC-5674) is strictly aerobic as it did not ferment carbohydrate (neither gas formation nor the color change) deep inside tire medium. Due to availability of oxygen on the surface of medium some color change was observed. Whereas £. coli grew very well deep inside the medium and fermented the carbohydrates (both gas formation and change in color of the medium) indicating that it is a facultative anaerobe (Fig. 8). In the negative control neither gas formation nor color change was observed,

2.1.1.7 Hydrogen Sulfide Production Test Material » SIM (Sulfide Indole Motility) medium * Culture tubes

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PCT/IN2015/000294 » 2?.oo// culture.* .Bacillus subtilis ssp, Wn'O/r/ensD (MTCC-5674) * Incubator

Method

The tubes containing SIM (Sulfide Indole Motility, {Annexure ~ I (Vii)}] .medium were labelled as “negative control·* and “test and a loop full of E. call and Bacillus subtilis ssp, s7?rr>w?eitt/i·· (MTCC-5674), were inoculated in the tubes respectively, and incubated at SOX for 24 hours and observed for color change.

Result

From the observations, it has been concluded tliat the test organism is negative for HjS production as the medium did not turn black. The same result was observed in the negative control (Fig. 9).

2-1.1.8 Effect of pH on the growth of Bacillus sub/ills ssp. shriramensis iMTCC5674)

Culture tubes containing standard culture medium (LB) adjusted to different pH values ranging from 3.4 to 11,0 (acidic to basic) were used to grow Bacillus subtilis ssp, shriramensis (MTCC-5674) under standard conditions. Growth of Baetffas· subtilis ssp, sfarjrawensfs (MTCC-5674) was observed in a pH range of 6,4 to 7,2 and the optimum pH was found to be 7,0.

2- L 1.9 Antibiotic- Sensitivity Test of Bacillus subtilis ssp. shriramensis (MTCC5674)

A 24 hours old Zlum'/his sub/i/A ssp. shriramensis (MTCC-5674} culture was spread over the surface of T3 agar, Different' antibiotic discs were placed on the surface of the T3 agar plates labelled with the respective antibiotic. The pistes were incubated at SOX for 24 hours.

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Result

Table» 1 Observations.....on.....antibiotic.

shriramensis (MTCC-5674) RES sensitivity of istant, ΪΝΤ

Bacillus subtilis ssp. - Intermediate. SEN -

s. No. / ?	Antibiotic	Code	Zone	Result	Standards
RES	INT	SEN
1	Ampieilhn	A10	0	RES	13	14-16	17
2	Gentamicin	G10	20	SEN	12	13-14	15
3	Tobramycin	'FBI a	15	SEN	12	13-14	15
4	Carbcniciilin	CB100	15	RES	19	20-22	23
5	Vancomycin	VA30	20	SEN	<15		15
6	Oxacillin	OXI	11	INT	10	11-12 .	13
7	Novobiocin	NV30	24	SEN	17	18-21	22
8	Sulfisoxazole	SF300	3.3	SEN	12	13-16	17
9	Amikacin	AK3Q	15	INT	14	15-16	’ 17
10	Kanamycin	RiO	13	RES	13	14-17	18
11	Streptomycin	sio	12	INT	Tt	12-14	15
12	Cephalothin	CH30	40	SEN	14	15-17	18
13	Chloramphenicol	¢30	25	SEN	12	13-17	18
14	Erythromycin	£15	16	INT	13	14-22	23
is	Enrofloxacin	EX 10	41	RES	N/A	: N/A	N/A
16	Li neomycin	L10	12	EES	N/A	N/A	N/A
17	Amoxicillin	AC30	0	RES	19		20
18	Clindamycin	CD2	10	RES	14	15-20	21
19	Ceftriaxone	030	28	SEN	13	14-20: ..... - - / -	21
20	Bacitracin	310	21	SEN	3	9-12	13
21	Neomycin	N30	12	RES ,1 12	13-16	17
22	Azithromycin	ATI 5	10	RES ) 13 I 14-17 - ...................i........................	18

It has been concluded from the observations that Bacillus subtiiis ssp. shriramensis (MTCC-5674) is resistant to the antibiotics - ampieilhn, carbenicillin, kanaraycin, enrofloxacin, iincotnycin, amoxicillin, clindamycin, neomycin, azithromycin. The test bacterium is sensitive to gentamicin, tobramiein, vancomicin, novobiocin, sulfisoxazole,

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PCT/IN2015/000294 cephalothin, chloramphenicol, ceftriaxone and bacitracin and showed intermediate resistance to oxacillin, amikacin, streptomycin and erythromycin..

2.1.2 identificalion of the.rtoy <j splate..Bgw/myjubri/A,ssp,x/?nrpme/tyfelM.TCC-5674)

For identification of bacteria, 16S DNA sequencing and FAME analysis were carried out. The results of both studies showed that the bacteria is showing 0.37% difference in 16S DNA. sequence and FAME similarity index of 0.827; with BacH/m sufai/;.? ssp. ,ί«Ζ?ζ7/β· and 0,84% difference in I6S DNA sequence and FAME similarity index of 0.749 with BadHas airophacwx Thus, the results indicate that this bacterium is related to Bac/Zte subd/Ls and RmriLW but not identical to any of the catalogued bacterial species in ATCC collection,

2.1.2.1 Results of 16S. DNA .sequence contparisop

Match	% Difference	Length	Library Entry Name
1 i.....................	0,19	535	Racttfus swbdVN ssp, s'ubh'/A
0	0,84	535	BaeiikiS iihmzzWm'
0	1.03	535	Bnctf/us· ΰηρνοΖή/ηΙ/ύΝοη,ν

2.1,2/2 Results of FAMEanalyslscpmnarNpn

S, No.	Similarity Index	Library .Entry Name
1	0,827	Buci/Mr iwbh/A
2	0,749

The isolated bacterium is a new member of the genus According to bacterial nomenclature convention, the novel bacterial species was named as B«£?h7«s i'ub/O'A ssp. ihrirninemA, The bacterium is deposited in the Microbial Type Culture Collection (MTCC) at IMTECH, Chandigarh, India. The deposition number of this novel species is (MTCC-5674).

Example 3

3.t Praducdpa,^d^ereenmg,<jhe.aminncrsbialand/br.andfepgalaggnt

3-1.1 tekictipnofanfimien^

Material * T3 broth - i L * Conical Flask - 2 L capacity ’22WO 2016/027279

PCT/IN2015/000294 » Kanamycin (30 pg/ml) » Bacillus subtilis ssp, shriratnertsis (MTCC-5674) inoculum » Shaking Incubator (set at 3O'^:’C temperature & 200 rpm shaking)

Method ' ' The T3 broth was prepared according the method described in Annexure - I (1), A 1 mi culture of Bacillus subtilis-ssp. shriramensis (MTCC-5674) was inoculated into the sterile T3 broth and incubated in the shaking incubator at 30°C for 60 hours, while shaking at 200 rpm. Followed by the growth of Bacillus subtilis ssp, shriramensis (MTCC-5674) in the T3 broth for 60 hours, the culture medium was centrifuged at 12000 rpm and 4°C for 10 min. The supernatant was collected and passed through 0,22 pm filters to separate out any residual bacterial cells. The filtrate was maintained at 4^QC.

3,1.2 Screening the antimicrobial and/or antifungal activity of the culture filtrate collected ttt step 3.1.1 above .Materia

Culture Filtrate of Bacillus subtilis ssp, shriramensis (MTCC-5674.) containing antimicrobial and/or antifungal agent PDA plates

Test fungus Fusarium oxysporum ' '

Incubator

Method To test the activity of antimicrobial and/or antifungal agent in fire filtrate, a well was made in one comer of the PDA agar plate, 500 pi of the filtrate was placed in the well. A loop full of the fungus FusaHum oxysporum was inoculated at the other corner in the same PDA agar plate and incubated for 5 days at room temperature, Inhibitory activities of the filtrate against the fungus Fusanum oxysporum were recorded as the inhibitory zone surrounding the well in millimetres.

Result

Clear inhibitory zone of 14 mm was observed surrounding the well, suggesting that the method used lor the production of antimicrobial and/or antifungal agent is optimum

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3-2 Charasterjzatlon of.antinaisiafeiaLsste

The antimicrobial and/or antifungal activity associated with Bacillus subiilis ssp, shriramensis was investigated to ascertain the nature of agent causing antimicrobial and/or antifungal activity,

Mgtgrtsl « T3 broth — 1 L • Conical Flask - 21., capacity * Kanamycin (30 pg/tni) ♦ Bacillus subiilis ssp, shriramertsis (MTCC-5674) inoculum * Shaking Incubator Method

The T3 broth was prepared according the method described in Annexure - I tl}, A 1 ml aliquot of 24 hours old Bacillus subiilis ssp, .yMrmnemfi (MTCC-5674) was inoculated into the sterile T3 broth and incubated in the shaking incubator at 30^vC for 60 hours, while shaking at 200 rpm. Following growth of &?e,7h,u· subiilis ssp, sAr/rmwcww (MTCC-5674) in the T3 broth for 60 hours, the culture medium was centrifuged at 12000 rpm and 4°C for 10 min. The supernatant was collected and passed through 0.22 um inters to remove any remaining bacterial cells.

3-2-1 Anthuleroblai, aud/OL.anil iMTCC-„55?4xbucterial.eei Is

To test the activity of antiinicrobial and/or antifungal agent by the cells, a loop hill of &m?7/n,v ,vwbi?/A ssp. .v/i.rt'?n?nensrt' (MTCC-5674) inoculated in one corner of the T3 agar plate and a loop full of the fungus busarium oxysporum was inoculated at the other corner in the same T3 agar plate and incubated for 5 days art room temperature, inhibitory activities of the bacterial cells against the fungus Fusarium oxysporum were recorded as the inhibitory zone surrounding the bacterial colony in millimetres,

Besuh

Clear inhibitory zone of 14 nun (Fig, 11 A) was observed surrounding the bacterial colony, suggesting that the active compound secreted by the bacterial cells is getting diffused out in the culture- medium resulting in clearance zone away from bacterial colony.

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3.2.2 Antimicrobial and/or antifungal assay with the Bacillus subtilis ssp. shriramensis (MTCC-5674) culture filtrate

To test the nature of antimicrobial and/or antifungal agent in the filtrate, a well was made in the PDA agar plate and 500 μΐ of the filtrate was placed in the well. A loop full of the fungus Fusarium oxysporum was inoculated at the diagonally opposite end of the same PDA agar plate and incubated for 5 days at room temperature. Inhibitory activities of the filtrate against the fungus Fusarium oxysporum were recorded as the inhibitory zone surrounding the well in millimetres.

Result

Clear inhibitory zone of 14 mm (Fig. 1 IB) was observed surrounding the well, suggesting that the filtrate retained antimicrobial and/or antifungal activity, thus indicating the active compound is secreted outside the bacterial cell in to the culture medium.

3.3 Determination of MIC of Bacillus subtilis ssp. shriramensis (MTCC-5674)· antimicrobial and/or antifungal agent

3.3.1 Lyophilization of antimicrobial and/or antifungal agent

Material • Culture filtrate of Bacillus subtilis ssp. shriramensis (MTCC-5674) • Ammonium sulphate • Freeze drier Method

The antimicrobial and/or antifungal agent was produced and purified by the methods explained in 3.1.1. A 800 ml aliquot culture filtrate was mixed with 382.18 g ammonium sulphate at 70% (w/v) saturation (modified protocol of Jing et al., 2009) and solution was gently mixed by stirring for overnight at 4°C. The suspension was centrifuged at 10,000 rpm for 10 min at 4°C. The pellet thus obtained was lyophilized for 24 hours in a freeze drier and the dried pellet was stored at room temperature.

3.3.2 MIC of Bacillus subtilis ssp. shriramensis (MTCC-5674) antimicrobial and/or antifungal agent

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Methods

3.3.2.1 Tube dilution method

3.3.2.2 Agar diffusion method jTeparahmi. of sipeLsofiM..^

The stock solution was prepared by dissolving 1. g of lyophilized powder of antimicrobial and/or antinmgai agent in SO mi phosphate buffer {pH 7.0). The final concentration of the stock solution was adjusted to 20 ug/nl, This stock solution was used for making dilutions with PDB media in different ratios as shown in Table-1. 3.3.2.1 Tube dihttipn.tnethpd

Material * i .5 ml rubes * PDB medium * Antimicrobial and/or antifungal agent stock * imsm-hon spore suspension

Method

MIC assay of antimicrobial and/or antifungal agent was carried out in 1.5 ml tubes. Different dilutions of antimicrobial and/or antifungal agent were prepared in PDB medium (Table -2) ranging from 10 pg/μΙ (IT) to 198 ng/pl (1:100). The MIC assay was carried out against Fmmnmn by adding 30 μΐ (5x11? cm/ml) of spore suspension in ail the tubes and were incubated at 28°C, for 2 days, shaking at 180 rpm.

Three controls were used, one with undiluted antimicrobial and/or antifungal agent stock, second with 70% ammonium sulfate in PDB and third with only PDB, Medium In all the three tubes was inoculated with 30 ul (5x10° cfc/nd) of Eusarwm oygroornv?? spores suspension and were incubated at 28°C, for 2 days, while .shaking at 180 rpm,

3-3.2,2 AgindiBustpn.tnethpd

M aterial ' * PDA (Potato Dextrose Agar) plates * PDB (Potato Dextrose Broth) medium ' * Antimicrobial and/or antifungal agent stock ' * /m.Viirimu oxp.$ywww '

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Method

MIC assay of lyophilized /DeZ/m's sub/zZ/s ssp. skrirametwte (MTCC-5674) antimicrobial and/or antifungal agent was also carried out by agar diffusion method. Four wells of 9 mm diameter each were made at equal distances in PDA plates. Different dilutions of antimicrobial and/or antifungal agent were prepared In PDB (Table-3) ranging from It) pg (1:1) to 198 ng (1:11)()). An aliquot 200 pi of each dilution was place in to die well labeled with the respective dilution. The test fungus Puswiiwi oxysponm was inoculated in the center of the FDA medium and the plates were incubated at 2tb'€ for 4 days, ’

A plate with three controls, one containing undiluted antimicrobial and/or antifungal agent stock, the second containing only PDB broth and the third containing PDB with 70 % ammonium sulphate was used as control, i'he activity was measured as inhibitory zone in millimeters surrounding the 'well. Result

MoRlutisM

The samples were observed after 48 hours of incubation under light microscope for spore germination. Spores did not germinate irt the tubes containing antimicrobial and/or antifungal agent in the ratios 1:1,1:2, 1:3 and 1:4. Moderate spore- germination was observed in the tubes containing antimicrobial and/or antifungal agent in the ratios 1:5, 1:6, 1:7, 3:8 and 1:9 dilutions, and normal spore germination and mycelia formation was observed in the remaining tubes containing the antimicrobial and/or antifungal agent in the ratios 1:10 to 1:100 (Tabled).

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Table>2 MIC of antimicrobial and/or antifungal agent by tube dilation method

S.No,	: Antimicrobial and/or antifungal agent; ΡΌΒ	Concentration of antimicrobial and/or tudifunga: agent after dilation (μ a/μΙ)	Total concentration of a n ti nticrobi a 1 and/or antifungal agent used per well in agar plate (tgCOQpi)	Percent of fungus inhibited (inhibition zone in mm)
1	1:1	10,001)	2000.00	92.30(12)
2	1:2.	6.666	1333.20	84.61 (H)
3	1:3	5.000	1000,00	76.92(10)
4	1:4	4.000	800.00	61.53(8)
5	1:5	3.333	666.60	46.15 <6)
6	1:6	2.857	571.40	38.46 (5)
7	1:7	2.500	500.00	23.07 (3)
8	1:8	2.222	444,40	15,.38(2)
9	1:9	2.000	400.00	0(0)
io	. ICO	1,818	363.60	0(0)
π	1:15.	1.250	250.00	0 (0)
12	1:20	0.952	190,40	0(0)
□	1:25	0.769	.153,80	0(0)
14	1:30	0,645	129.00	0(0)
15	1:35	0.555	Π I.0O	0(0)
16	1:40	0.487	97,40	0(0)
17	1:45	0,434	86,80	0(0)
18	1:50 .	0.392	78.40	0(0)
.19	1:55	0.35?	71.40	0(0)
20	1:60	0.32'?	65.40	0 (0)
21	1:65	0,303	60.60	0 CO)
22	1:70	0,28 Ϊ	56,20	0 (0)
23	1:75	0,263	52.60	0(0)
24	1:80	0.246	49.20	' 0(0.)
25	1:85	0,232	46.40	0(0)
26	i :90	0,219	43,80	0 (0)
27	1:95	0.208	41.60	0 (0)
28	1:100	0.198	39.60	0(0)
29	Only antimicrobial and/or antifungal agent	Crude (20 pg/μΙ)	4000.00	100 (13)
30	OnivPDB medium	0	0	0(0)
31	Only ammonium : sulphate 70%	0	0	0 t 0)

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Agar diffusion.method (Fig· 13) inhibition of fungal mycelium growth was observed around the wells containing antimicrobial and/or antifungal agent in the ratios 1:1, 1:2, 1:3 and 1:4 (Table 3). Moderate inhibition was observed surrounding the wells containing antimicrobial and/or antifungal agent in the ratios 1:5, 1:6 and 1:7 dilutions and no inhibition were observed in the remaining dilutions (from 1:8 to 1:100) (Table 3).

Conclusion

From the above experiment it is concluded that the antimicrobial and/or antifungal agent in powder of crude extract is inhibiting spore germination as well as mycelium growth upto a dilution of 1:4 (v/v), in a concentration dependent manner, >23'

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Table-3 MIC of antimicrobial and/or antifungal agent by agar diffusion method

S.No.	Antimictobial and/or antifungal agent: PD8	Concentration of antimicrobial and/or antifungal agent after dilution Will)	Total CQncentration of antimicrobial and/or antifungal agent used per tube (gg/200pi)	Spore germination
1	in	10,000	2000,00	No germination
2	1:2	6,666	1333.20	No germination
3	1:3	5.000	1000.00	No germination
4	1:4	4.000	800,00	No germination
5	1:5	3,333	666.60	Germ tube emergence
6	1:6	2,857	571.40	Germ lube, growth
7	1:7	2,500	500.00	Germ tube elongation
8 ’	1:5?	2,222	444.40	Myceiia growth
9	1:9	2,000	400.00	Myceiia extension
10	1:10	1,818	363.60	Compact mycelia
n	1:15	1,250	250,.00	Compact myceiia
12	1:20	0.952	190,40	Compact mycelia
13	1:25	0,769	153.80	Compact mycelia
14	1:30	' 0.645	129.00	Compact mycelia
IS 1 1:35	0.555	111.00	Compact myceiia
16	1:40	0.487	97.40	Compact mycelia
17	1:45	0,434	86,80	Compact myceiia
18	1:50	0.392	78,40	Compact mycelia
19	i:S5	0.357	71,40	Compact mycelia
20	1:60	0-327	65.40	Compact mycelia
21	1:65	0,303	60,60	Compact mycelia
22	1:70	0,281	56,20	Compact mycelia
23	1:75	0.263	52.60	Compact mycelia
24	1:80	0,246	49.20
25	1:85	0.232	46.40	Compact mycelia
26	1:90	0,219	43.80	Compact mycelia
27	1:95	0.208	41,60	Compact mycelia
28	1:100	0.198	39.60	Compact mycelia
29	Only antimicrobial and/or antifungal agent	Crude(20 pg/ul)	4000,00	No germination
30	Only PDB	0	0	Compact mycelia
3i	Only ammonium sulphate 70¾	0	0	Compact mycelia

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3-4

M&Misl .

* .PDA agar plates » LB Broth * Bacillus ssp. Miwmnmw (MTCC-5674) cells * Lysozyme * Incubator

Methods

....... _: ^: - : :

3.44

The LB broth was prepared according to the method described in Annexure I (3). A single colony of Bne/Aws suofi/B ssp. s/mirumeiisfi· (MTCC-5674) was inoculated into the sterile T..B broth and Incubated at 30^yC for 24 hours. After 24 hours of incubation : the vegetative celts were collected by centrifugation at 6500 rpm, 4°C, the ceils were washed thrice with sterile distilled water and subjected to cell lysis by incubation in lysozyme at eV'-'C for 2 h. After lysis the suspension was centrifuged at. 1(),000 rpm for 10 min at 4^0 to remove the cell debris. The supernatant was collected and passed through 0.22 urn filter and stored at 4° C.

3-^{3 4}2 Antinucrpblai andfor.amuungalj^^

Two wells were bored in two diagonal ends of the PDA agar in the petri plate and labelled one as “test” and the other as “control’·’. An aliquot of 500 p.1 of the lysate was added to the test well and 500 μΐ of only lysozyme was added to the control well. The test fungus Faswr/.w?? arysporwn was inoculated in the middle of the PDA agar and. Incubated for 5 days at room temperature, tom

The cell lysate did not exhibit antimicrobial and/or antifungal activity (fig, 15) against, the fungus Fusarium ayt-rfum-mn, suggesting that the antimicrobial and/or antifungal agent is primarily secreted out into the media.

Example 4

To test the antimicrobial and/or antifungal activity against other pathogenic fungi

Material » Bari/lus subiilis ssp. shriramenris (.MTCC-5674) ’ - 31 WO 2016/027279

PCT/IN2015/000294 * Plant Pathogenic fungi

1. Jitiizadawa solan; (Causes sheath blight in members of family soianaeeap

2. Saraeladfum orjtzoe (Causes sheath rot in rice)

3. Coileronic/aaa oupstcii (Causes anthracnose in chilli.).

I4, £xerafa1wn turcicum (Causes turcicum blight),

5, MacfOphomma phaseottna (Causes charcoal rot).

T3

- broth * T3 Agar plates * PDA - Agar plates * Incubator MaihpJ

43.1 Is.teaiheanthnierpbiai and/M^)ote

A loop full of Bacillus subfills ssp. shrinwictisis (MTCC-5674) cells and a loop full of test fungi were inoculated at. the diagonally opposite ends of the T3 plates labeled

I with the respective fungus and incubated at 28°C till the growth of fungal mycelium was observed in the vicinity of the bacterial colony.

4.1.2 JSLUgnhiL^Jlnmbid^S^^

An aliquot of 500 μΐ of the culture filtrate containing antimicrobial and/or antifungal agent was added Into the wells made in the PDA agar and a loop full of test fungi were inoculated at the other corner of the respective plates labeled with, the respective fungus and incubated at 28^eC till the growth of fungal mycelium was observed in the vicinity of the well containing culture .filtrate.

I The inhibitory activity of the filtrate against the target fungus was recorded in I millimetres as the inhibitory zone formed surrounding the well.

teult

A range of fungal species causing diseases in plants were tested in the antimicrobial and/or antifungal assay and all of them demonstrated complete inhibition of growth in the presence of Bacillus subtilis ssp, shNratacasls (MTCC-5674) cells and also its culture filtrate.

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4.2 Efficacy of antimicrobial and/or antifungal agent in protecting rice seed from fungal attack

Rice seeds were treated with Fusarium oxysporum spores and Bacillus subtilis ssp. shriramensis (MTCC-5674) culture filtrate and placed in the petri plates containing plain agar to check the efficacy of Bacillus subtilis ssp. shriramensis (MTCC-5674) antimicrobial and/or antifungal agent in inhibiting the fungal attack on germinating seed.

Control seeds were treated only with Fusarium oxysporum fungal spores.

Result

In presence of Bacillus subtilis ssp. shriramensis (MTCC-5674) antimicrobial and/or antifungal agent fungus failed to infect the seeds and the rice seeds germinated normally. However, the seeds treated only with fungus showed severe infection and failed to germinate (Fig. 17).

4.3 To test the pathogenic nature of Bacillus subtilis ssp, shriramensis (MTCC-5674) of plants '

Material ' • Bacillus subtilis ssp. shriramensis (MTCC-5674) in 1 % CMC in sprayable form • Rice, cotton, tobacco, com and tomato plants • Sprayer

Method .

The Bacillus subtilis ssp. shriramensis (MTCC-5674) culture was extensively tested for pathogenic behaviour if any, on a range of plant species.

Bacillus subtilis ssp. shriramensis (MTCC-5674) was inoculated into 1 L sterile LB broth in a 2 L conical flask and incubated at 30°C, for 24 hours, shaking at 200 rpm. Following the growth of the bacteria, the cells were harvested by centrifuging at 6,500 rpm, at 4°C for 10 min. The pellet was washed twice in phosphate buffer (pH 7.0) and made into slurry in 1% CMC (Carboxy Methyl Cellulose) in phosphate buffer (pH 7.0). This suspension was used for spraying on crop plants like rice, tobacco, com, tomato and cotton.

Result

From the observations it has been concluded that, all the plant species (rice, tobacco, com, tomato and cotton) sprayed with Bacillus subtilis ssp. shriramensis (MTCC-33WO 2016/027279

PCT/IN2015/000294 : 5674) did not exhibit any kind of disease symptoms and their growth and development was equivalent to control plants indicating that Bacillus subtilis ssp. shriramensis (MTCC-5674) is non-pathogenie for plant species (Fig. 18).

Example 5

5.1 Formulation of antimicrobial and/or antifungal compositions containing Bacillus subiilis ssp. shriramensis (MTCC-5674) cells as a biological control agent Material » Bacillus subiilis ssp. shriramensis (MTCC-5674) * LB - broth * FDB (Potato Dextrose Etroth) » Phosphate buffer (pH 7.0) * CMC (Carboxy Methyl Cellulose)

Method

5. LI Preparation of BrmB/ax subtilis ssp. shriramensis (MTCC-5674) cell suspension

Bacillus subiilis ssp. shriramensis (MTCC-5674) was inoculated in 1 L sterile LB broth in a 2 L conical flask and incubated at 30°C, for 24 hours, shaking at 200 rpm. Following the growth Bacillus subtilis ssp, shriramensis (MTCC-5674), the culture was centrifuged at 6,500 rpm, at 4 °C for 10 min. The pellet was washed twice in phosphate buffer (pH 7,0) and mixed with 1% CMC (Carboxy Methyl Cellulose) in phosphate buffer (pH 7.0) io prepare a slurry containing 6x10' efu/mi. The slurry containing Bacillus subtilis ssp, shriramensis (MTCC-5674) was used to spray on plants and treat plant seedling roofs by dipping,

5.1.2 To test the efficacy of formulate containing antimicrobial and/or antifungal agent to inhibit the infestation of Bhisacionia salani (NPCCI-3194) in the roots of tomato

Material

Slum containing Bacillus subiilis ssp, shriramensis (MTCC-5674)

Rhiztfciania salani (NFCCI-3194) fungus (causes sheath blight in members of family soianacea),

Soil rite

Tomato seedlings

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5.1.3 Preparation οι Rhizoctonia solani (NFCCI-3194)

Rhizoctonia solani (NFCCI-3194) was grown in Potato Dextrose Broth (prepared as per the method provided in the Annexure - I (V) medium for 6 days. Following the growth of the Rhizoctonia solani (NFCCI-3194). it was thoroughly mixed with soil rite and incubated for 15 days at room temperature. The soil rite containing the fungus was mixed with soil in 1:1 ratio.

• Tomato seedlings

Tomato seedlings of 10 cm height were used in this study Method

The experiment was carried out as described below

A. Tomato seedlings were planted in the soil containing Rhizoctonia solani (NFCCI3194). but were not treated with Bacillus subtilis ssp. shriramensis (MTCC-5674).

B. The roots of Tomato seedlings were treated with slurry containing Bacillus subtilis ssp. shriramensis (MTCC-5674) and were planted in the soil containing Rhizoctonia solani (NFCCI-3194).

C. Tomato seedlings without any treatment. '

A. Seedling treatment with Bacillus subtilis ssp. shriramensis (MTCC-5674) cells and fungus Rhizoctonia solani (NFCCI-3194)

Tomato seedling roots were dipped in the Bacillus subtilis ssp. shriramensis (MTCC5674) cell formulate for 30 min. The treated seedlings were planted in the pot containing soil mixed with the fungus Rhizoctonia solani (NFCCI-3194).

Control seedlings ‘

For inducing the disease in the seedlings, tomato seedlings (untreated) were planted in the pot containing soil mixed with the fungus Rhizoctonia solani (NFCCI-3194).

Tor negative control tomato seedlings (untreated) were planted in pot containing soil which is not mixed with the fungus Rhizoctonia solani (NFCCI-3194).

All the pots containing tomato seedlings were transferred to the green house and maintained till fruiting stage.

Result

From the observations it was concluded that the seedlings treated with combination of Bacillus subtilis ssp. shriramensis (MTCC-5674) and the fungus Rhizoctonia solani (NFCCI-3194) grew very well equivalent to control plants, whereas the seedlings

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PCT/IN2015/000294 (untreated) planted in the pot containing fungus Jihizocfonia salani (NFCC1-3194) exhibited retarded growth, poor flowering and fruit formation as compared with control, Hence, it can been concluded that Bacil/w .wfe/fe ssp, shriramensis· (MTCC5674) inhibited the growth of the fungus Xhizoetonia so/mn (NFCCI-3194) in the rhizosphere area of tomato seedlings and protected the seedlings from disease causing fungus (Fig. 19).

Example 6

6fl £?.rimo.m£lnafion.cf mmimum^

64,.1 Preparation of bacterial and fungal suspension cultures Materials

a. Ztoetf/us .mbfl/A ssp. s/?rfl«/nens&· (MTCC-5674)

b. FenicBBum axalician (NFCCI-1997)- plant pathogenic fungus

c. Carbendazim WP50 (commercial fungicide)

d. Turin Bertani Broth (LB)

Method

64.1,1 Bmnamfion,,o,r,su$gen^...cuftum £MTCC5674)

A pure colony of Z?act7/i«· WmVA ssp, shriramensis (MTCC-5674) (Fig. 20-1) was inoculated In 10 ml LB broth and incubated at 30°C for 24 h at ISO rpm. For preparation of bio-control formulation 1 mi of flesh culture was inoculated in 100 ml of LB broth and incubated at 3G*C for 24 h at 180 rpm. Growth of culture was monitored by periodic measurement of absorbance of culture at 625 nm. The bacterial cells were harvested by

I centrifugation and washed with sterile phosphate buffer, by centrifugation at 5500 rpm for 10 min at 4^f'C. The cells were finally suspended in 5 ml of sterile phosphate buffer. This concentrated suspension was used, for preparation of bio-control formulations.

6.1.1.2 Preparation.of ffiingaUidbPgen)

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Pure colony of Penicillium oxalicum (NFCCI-1997) (Fig. 20-2) was inoculated on PDA plate and incubated at 28°C till spore formation. A loop full of the fungal spores were inoculated in 100 ml of PDB and incubated at 28°C for 7 days at 180 rpm. The aqueous part of the culture containing fungal spores was collected in 50 ml polypropylene tubes. The spores were washed with sterile phosphate buffer by centrifugation at 8000 rpm for 10 min at 4°C. The spores were suspended in required volume of sterile phosphate buffer to obtain a cfu of 6x10⁴ ml¹.

6.1.2 Soil infestation with P, oxalicum (NFCCI-1997) (fungal pathogen)

To maintain adequate fungal spore load in the soil medium, 50 ml fungal spore suspension (6xl0⁴ cfu/ml) was mixed with 1 kg of autoclaved soilrite and incubated for 10 days at 28°C. The soilrite colonized with fungus was uniformly mixed with soil in 1:1 ratio and filled in 96 cup trays.

6.1.3 Preparation of formulation of Bacillus subtilis. ssp, shriramensis (MTCC-5674) for biological control of soil borne plant disease

Material

a. CMC (Carboxy Methyl Cellulose)

b. Sucrose

c. Red polymer (without fungicide)

d. Bacillus subtilis ssp. shriramensis (MTCC-5674) cell suspension (bio-control agent)

e. Carbendazim (commercial fungicide)

To assess effective concentration of Bacillus subtilis ssp. shriramensis (MTCC5674) cells Which can suppress growth and pathogenicity of P. oxalicum (NFCCI1997) on germinating com seed, four different formulations were designed (details are provided in table below). Formulations containing only bio-control agent, only commercial fungicide, and one without bio-control agent or fungicide were used as controls. All the formulations contain a binding material - CMC (Carboxy Methyl Cellulose), carbon source (sucrose) and a red polymer (without fungicide).

1. Control-1 (Formulation without fungal pathogen and bio-control agent)

This formulation is composed of 1% CMC, 2% sucrose, and fed polymer. This formulation has no bio-control agent, disease causing agents and fungicide. Seeds treated with this formulation were used as control seeds.

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Composition

S.No.	Components	Weight/V olume/Number	Final Concentration
1	CMC	1.71 pg	1.00 % w/v
2	Sucrose	. 3.42 pg	2.00 % w/v
3	Red Polymer	34 μΐ	19.88 %v/v
4	Water	31.87 pi	' -
	Total	171 μΐ	-

2. Control-2 (Formulation with fungal pathogen but no bio-control agent)

This formulation is composed of 1% CMC, 2% sucrose, and red polymer. It has no bio-control agent/commerclal fungicide, but the seeds treated with this formulation were sown in the soil inoculated with P. oxalicum (NFCCI-1997) fungus. As there is no biological or chemical protection around the seeds, the fungus grows profusely, infects the seeds and develops disease in the seedlings. The seeds treated with this formulation are used as diseased controls.

Composition

S.No.	Components	Weight/Volume/ Number	Final concentration
1	P. oxalicum (NFCCI-1997)	Present in the soil	-
2	CMC	1.71 pg	1.00 % w/v
3	Sucrose	3.42 pg	2.00% w/v
4	Red Polymer	34 pi	19.88% v/v
5	Water	31.87 pi	-
	Total	171 pi	-

3. Control-3 (Formulation with commercial fungicide “Carbendazim WP50”) .

This formulation is composed of 1% CMC, 2% sucrose, red polymer and a commercial fungicide Carbendazim WP50 (trade name Bavistin) was used at a concentration of 500 pg/ml (Mohiddin et al., 2013). This formulation is used to compare the efficacies of both bio-control agent and the commercial fungicide in suppressing the fungal growth in the vicinity of the germinating seed.

Composition

S.No.	Components	Weight/Volume/ Number	Final concentration
1	Carbendazim	85.50 pg
2	CMC ·	1.71 pg	1.00 % w/v
3	Sucrose	3.42 pg	2.00 % w/v

•38'

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4	Red Polymer	34 μΐ	19.88% v/v
5	Water	31.87 μΐ	-
	Total	171 μΐ	-

4a. Formulation with Bacillus subtilis ssp, shriramensis (MTCC-5674) (5x10⁴ cfu)

This formulation is composed of 1% CMC, 2% sucrose, red polymer and Bacillus subtilis ssp. shriramensis (MTCC-5674) cells at a concentration of 5xl0⁴ cfu/ml. This formulation has minimum number of Bacillus subtilis ssp. shriramensis (MTCC-5674) cells.

Composition

S.No.	Components	Weight/Volume/ Number	Final concentration
1	Bacillus subtilis ssp. shriramensis (MTCC-5674) cell suspension	100.00 μΐ	5x104 cfu/ml
2	CMC	1.71 μβ	1.00 % w/v
3	Sucrose	3.42 qg	2.00% w/v
4	Red Polymer	34.0 μΐ	19.88 % v/v
5	Water	31.87 μΐ	-
	Total	171.0 μΐ	-

4b. Formulation with Bacillus subtilis ssp, shriramensis (MTCC-5674) cells (5x10^s cfu)

This formulation is composed of 1% CMC, 2% sucrose, red polymer and Bacillus subtilis ssp. shriramensis (MTCC-5674) cells at a concentration of 5x10^s cfu/ml. Composition

S.No.	Components	Weight/Volume /Number	Final concentration
1	Bacillus subtilis ssp. shriramensis (MTCC-5674) cell suspension	100.00 μΐ	5x10s cfu/ml
2	CMC	1.71 gg	1.00 % w/v
3	Sucrose	3.42 μg	2.00 % w/v
4	Red Polymer	34 μΐ	19.88% v/v
5	Water	31.87 μΐ	-
	Total	171 μΐ	-

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4e.

This formulation is composed of 3% CMC, 2% sucrose, red polymer and #«<?//&«? subsiliy ssp. shrirumensis (MTCC-5674) cells al a concentration of 5x10^{5 6}cfu/ml. Composition

S, No.	Components	Weight/V olume/ Number	Final concentration
1	BncEhfs· vnN.Es' ssp, s'E/-z><??nezzsB (MTCC-5e?4) ceil suspension	100.00 pi	5xI0e eih/ml
2	CMC	. 1-71 pg	1.00% w/v
3	Sucrose	3,42 pg	2.00 % w/v
4	Red Polymer	34 pi	19.88 % v/v
5	Water	31.87 μΐ	-
	'Total	171 μϊ	-

4d. FbrmyissmnjsMtolsgjtegs^^

This formulation is composed of .1% CMC, 2% sucrose, red polymer and BneE/nx χηΡΕ/Β ssp. xEr/rmuemB (MTCC-5674) ceils at a concentration of 5xl0⁷cfo/mi (50 million cells/ml of carrier). This formuiation has maximum number of Baeiitw xzxNBff ssp, sMwh$ (MTCC4674) e-ells.

Composition

S.No.	Components	Weight/Volume /Number	Final concentration
I	BnczE/nx χηΜΕ,? ssp. (MTCC-5674) cell suspension	100.00 pi	5x1 (f efb/ml
2	CMC	1.71 pg	TOO % w/v
3	Sucrose	3.42 pg	2.00 % w/v
4	Red Polymer	34 pi	19.88 % v/v
5	Water	31,87 pi
	Total	171 p|

5. E9rmnlatimmMth_pm3yBopi/Ms,¥n0h/^ efo)

OCX', .'...W

This formulation is composed of 1% CMC, 2% sucrose, red polymer and BtreE/ns szib/EC ssp. shriramensis (MTCC-5674) cells at a concentration of 5x10' cfu/ml. This formulation has maximum number of Bcrni/m ssp.

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PCT/IN2015/000294 shriramensis(MTCC-5674) cells and is used to study the effect of biocontrol agent on seed germination and plant growth. Composition

S.No.	Components	Weight/Volume /Number	Final Concentration
1	Bacillus subtilis ssp. shriramensis (MTCC-5674) cell suspension	100.00 μΐ	5X107cfu/ml
2	CMC	1.71 pg	1.00% w/v
3	Sucrose	3.42 pg	2.00 % w/v
4	Red Polymer	34 pi	19.88% v/v
5	Water	31.87 pi	-
	Total	171 pi	-

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Table-4 Experiment plan in tabular form

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PCT/IN2015/000294

	20	20
	20	o
	20	20
	Nil	Nil
	S £	Nil
	O	t-x o V)
(Concentration-3)	Com seed + Red polymer + CMC + Sucrose + Bacillus subtilis ssp. shriramensis (MTCC-5674) cells (Concentration-4)	Com seed + Red polymer + CMC + Sucrose + Bacillus subtilis ssp. shriramensis (MTCC-5674) cells
	5 *

Ο

#

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Seed coating

The bio-control formulations as per the compositions given above were coated on com seeds (Fig. 21). Twenty com seeds in triplicates (total 60 seeds) for each treatment were surface sterilized with 0.1% HgCb for 10 min and rinsed with 95% ethanol, and washed with sterilized water for 10 min each. Dry seeds were coated with 171 μ1/60 seeds of different formulations and air dried for 2 h.

Seed sowing

All the treated seeds were sown in 96 cup trays with three replicates per treatment. All trays were kept in glasshouse and maintained under controlled conditions. From seed germination onwards the trays were monitored till 5 weeks.

Data Recording

Germination percentage

Germination percentage of all the seed treatments was recorded after 1 week of seed sowing.

Disease Incidence

Disease incidence was recorded as percentage after 4 weeks of seed sowing. The formula used for recording disease incidence (Hoffman et al., 2002) is as follows:

No. of diseased seedlings % disease incidence = - X100

Total No. of seedlings

Results

Seed germination and seedling survival

Optimum seed germination i.e., 93.33%, 96.66%, 100.00%, 100.00%, 100.00% and 100.00% was recorded in seeds treated with formulations - 3(control-3), 4c, l(control-l), 4b, 4d and 5, respectively, followed by 83.33% 33.33% in the seeds treated with formulations - 4a and 2(control-2). The seedling survival rate after 4 weeks of sowing was recorded as 100.00 % in the seeds treated with all the formulations mentioned above except in the seeds treated with the formulation-3 (which has commercial fungicide), this clearly indicates that the commercial fungicide “Carbendazim WP50”, though it was efficient in suppressing fungal growth, but was not 100.00 % efficient. The formulations containing different concentrations (except formulation 4a- which has least number of cells) of Bacillus . 44 ' ~ WO 2016/027279

PCT/IN2015/000294 subtilis ssp. shriramensis (MTCC-5674) cells proved to be 100.00 % effective in protecting seeds from P. oxalicum (NFCCI-1997) present in the soil.

The drop in germination rate of seeds treated with formulation - 4a (formulation with least concentration of bacterial cells, 50,000 cells/ml carrier) is clear indication that a basal dose of bacterial cells is required to confer protection to the germinating seeds against P. oxalicum (NFCCI-1997) present in die soil. Thus, formulation-4b, which has a bacterial concentration of 5x10^s cfu/ml (0.5 million cells/ml) conferred good protection against P. oxalicum (NFCCI-1997) and gave 100% seed germination and seedling survival rate, same as control seeds.

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PCT/IN2015/000294 ω

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Percentage variation (Values in %)	©	-100	© 1	n 00 1	©	©	©	©
Data recorded after 4 weeks of sowing		©	©	©	©	©	©	o	ό
O .S Έ 3 s S3 *U Λ w & sp O *0 2 o 123 *S o'- 'Z ο Ό δ O j5 S, « o o	©	©	o	5 (10%)	©	©	©	©
No. of seedlings survived (Values in %)	60(100%)	©	54(90%)	9 (18%)	| 60 (100%)	59(100%)	(%00l) 09	60 (100%)
Data recorded after 2 weeks of	Uh 3) ? £ -g j a J £ £ IP 1 i £ -a V)	©	©	©	©	©	©	©	©
Uh S) ® » *a Sis 1 § §<? 2 rpf	©	20(100 %)	2(3.33%)	1 36 (72%)	©	©	©	©
No. of seedlings survived (Values in%)	(%00l) 09	0(0%)	54 (96.42% )	1 Η (28%)	60 (100%)	59 (100%)	60 (100%)	60 (100%)
No. of seed germinated (Values iu %)	60(100%)	20(33.33%)	56(93.33%)	50 (83.33%)	60(100%)	59(96.66%)	60(100%)	60(100%)
ΐ ° 1 I .a v> & *3	©	© Ό	§	o so	© ©	© VO	© SO	© so
Concentratio nof Carbendazi m(gg)	t	1	85.50 I	1	1	1	1	1
Concentration of P. oxalicum (NFCCI-1997) (cfu/ml)	<	© w·* v£	© VO	S VO	©	© Ί* Ό	O VO	1
Concentration of Bacillus subtilis ssp. shriramensis (MTCC5674) cells (cfu/g) I 1	1	1	1	© 75 «Λ	© ** m	© X w-i	© 75	Γ* © ** m
Treatment	*	* rs	* _	4a*	4b*	4c*	# 5	♦

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Disease incidence

The results of the study showed that there was significant difference between the treatments. The seeds treated with the formulations 1, 4b, 4c, 4d and 5 did not exhibit any disease symptoms and displayed healthy growth, similar to control seedlings, indicating that the biocontrol agent Bacillus subtilis ssp. shriramensis (MTCC-5674) present in the formulations greatly suppressed growth and pathogenicity of the fungus P. oxalicum (NFCCI-1997), and thus protected the seeds from getting infected with the fungus. The seeds treated with the formulation-3 (which has a commercial fungicide Carbendazim 50WP) showed a disease incidence of 3.57 % indicating that though the commercial fungicide was effective in suppressing the fungal growth, but not as good as bio-control agent used in this study.

Table-6 Percentage disease Incidence of com seedlings treated with different formulations.

Treatment	Concentration of bacterial cells	Concentration of fungal spores in soil	Total No. of seedlings	No. of diseased seedlings	Percent Disease incidence
1 (control-1)	Nil	Nil	60	0	0.00
2(control-2)	Nil	6x104	20	20	100.00
3(control-3)	Nil ·	6xl04	56	2	3.57
4a	5x104	6x10*	50	41	82.00
4b	5x10s	6x104	60	0	0.00
4c	5xl06	6xl04	59	0	0.00
4d	5x107	6xl04	60	0	0.00
5	5x107	Nil	60	0	0.00

Disease incidence of 82 % and 100 % was recorded in the seeds treated with the formulations 4a and 2, respectively. The results indicate that the Bacillus subtilis ssp. shriramensis (MTCC-5674) cell density present in the formulation 4a was not effective in suppressing growth of fungus and hence the germinating seeds were infected with the fimgus and died after 2 weeks of germination. As expected, the seeds treated with formulation -2 which has neither bio-control agent nor commercial fungicide showed 100% disease incidence indicating that the fungus infected the germinating seeds and killed the seedlings within 2 weeks of germination.

Conclusion

The above results clearly indicate that Bacillus subtilis ssp. shriramensis (MTCC-5674) at a concentration of 5x10^s cfu/ml (formulation-4b) is effective in suppressing growth of fungal pathogen and gives 100% protection to germinating seedlings of com. Hence, the bio48

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6.2 Testing the efficacy of the Bacillus subtilis ssp, shriramensis (MTCC-5674) cells formulation in promoting the growth and yield in plants

Materials and methods

Materials

1. Seeds of Com, Tomato and Brinjal treated with the formulation as mentioned in 6.1.3 (4b).

2. Control seeds of Com, Tomato and Brinjal.

Methods

Seed coating

Seeds of Corn, Tomato and Brinjal were treated with the formulation mentioned in 6.1.3 (4b) and air dried.

Seed sowing

Treated and untreated (control) seeds of Com, Tomato and Brinjal, each in three replicates and each replicate containing 23 seeds were sown in the field of 4 meters area. The standard spacing measurements like 20 cm plant to plant and 60 cm row to row distance were maintained. Appropriate agronomy practices were followed to grow these crops to maturity.

Results

There were significant increase in plant growth parameters and yield under field conditions. The seeds coated with formulate containing Bacillus subtilis ssp. shriramensis (MTCC-5674) cells increased the yield in Com, Brinjal and Tomato by 17.60, 37.15 and 1.58%, respectively (Table -7). The Com, Brinjal and Tomato plants showed higher rate of growth, development and biomass accumulation (a representative picture of difference in treated and untreated Com is given in Fig 25). Earlier reports on plant growth promoters have also proved that the formulation containing Bacillus subtilis enhanced the growth of plants and induced systemic resistance to disease protection by producing 60 different types of secondary metabolites (Compant et al., 2005 and Mohan Kumar et al., 2015).

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Table- 7 Effect of Bacillus subtilis ssp. shriramensis (MTCC-5674) cells formulation on the yield of Com, Tomato and Brinjal.

S.No.	Crop	Treatment	Yield
1	Com	1% CMC, 2% sucrose, red polymer and Bacillus subtilis ssp. shrirarhensis (MTCC-5674)cells at a concentration of 5x10s cfu/ml (0.5 million cells/ml of carrier)	233.7
		Control	198.72’
		Increase over control	17.60%
2	Brinjal	1% CMC, 2% sucrose, red polymer and Bacillus subtilis ssp. shriramensis (MTCC-5674)cells at a concentration of 5x10s cfu/ml (0.5 million cells/ml of carrier)	727
		Control	457
		Increase over control	37.15%
3	Tomato	1% CMC, 2% sucrose, red polymer and Bacillus subtilis ssp. shriramensis (MTCC-5674)cells at a concentration of 5x10s cfu/ml (0.5 million cells/ml of carrier)	860
		Control	847
		Increase over control	1.58%

Example 7

7.1 Screening the efficacy of antifungal/antimicrobial agent to inhibit the growth of human pathogenic fungi

A range of fungal species causing diseases in human beings were isolated from the people suffering from various skin and lung infections. The antifungal and/or antimicrobial activity was tested against all the isolated human pathogenic fungi. Materials and Methods

Materials

1. Penicillium ssp.

2. Aspergillus flavus ’

3. Aspergillus niger

4. Aspergillus nidulans

5. PDA plates

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6. Antifungal/antimicrobial agent isolated from Bacillus subiilis ssp, shriramensis (MTCC-5674)

Method

An aliquot of 500 μΐ of the culture filtrate containing antimicrobial and/or antifungal agent was added into the wells made in the PDA agar and a loop full of test fungi were inoculated at the other comer of the respective plates labeled with the respective fungus and incubated at 28°C till the growth of fungal mycelium was observed in the vicinity of the well containing culture filtrate.

The inhibitory activity of the filtrate against the target fungus was recorded in millimetres as the inhibitory zone formed surrounding the well.

Result

A range of fungal species causing diseases in human beings were isolated from the people suffering from various skin and lung infections tested in the antimicrobial and/or antifungal assay and all of them demonstrated complete inhibition of growth in the presence of Bacillus subiilis ssp. shriramensis (MTCC-5674) culture filtrate (Fig. 24). Conclusion

From the observations it has been concluded that the Antifungal/antimicrobial agent isolated from Bacillus subtilis ssp. shriramensis (MTCC-5674) can be used in pharmaceutical applications also.

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References

1. Compant , S., Duffy, B., Nowak, J., Clement, C. and Barka, E.A. (2005). Use of plants growth promoting bacteria for biocontrol of plant diseases: Principles, Mechanisms of action, and future prospects. Appl. Environ. Microbiol. 71:4951-4959. Sci. World J., Vol 2012, pp. 001-012.

2. Hoffmann, W.A. and Poorter, H. 2002. Avoiding Bias in Calculations of Relative Growth

Rate. Ann. Bot., Vol.90 (1), pp. 37-42. .

3. Li, J. Yang, Q. Zhao, L-H, Zhang, S.M., Wang, Y.X. Xiao-yu and Zhao, X.Y. 2009. Purification and characterization of a novel antifungal protein from Bacillus subtilis strain B29. J. Zhejiang Univ. Sci. B.,Vol. 10 (4) pp. 264-272.

4. Malusa, E. Sas-Paszt, L. and Ciesielska, J. 2012. Mena-Violante, H.Cr. and Olalde-Portugal, V. 2007. Alteration of tomato fruit quality by root inoculation with plant growth-promoting rhizobacteria (PGPR): Bacillus subtilis BEB-13bs. Sci. Hort.,Vol.l (113), pp. 103-106.

6. Mohan Kumar, S.P., Chowdappa, P. and Krishna, V. (2015). Development of seed coating formulation using consortium of Bacillus subtilis OTPB1 and Trichoderma harzianum OTPB3 for plant growth promotion and induction of systemic resistance in field and horticultural crops. Indian Phytopath. 68 (1):25-31.

7. Mohiddin, F. A. and Khan, M. R. 2013. Tolerance of fungal and bacterial bio-control agents to six pesticides commonly used in the control of soil borrie plant pathogens. Global J. Pests, Dis. Crop Prot., Vol. 1 (1), pp. 001-004.

Relevant Patents ^:

1. A novel strain of Bacillus for controlling plant diseases and com rootwoim. (EP981540A1).

2. Strain of Bacillus subtilis for agricultural use. (W02009031874A1).

3. Antifungal Bacillus subtilis and a microorganism wettable powder containing the same (KR2011075132A).

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Aaagtrc I

Connwriilpnof Culture Media used

Note; A general method of media preparation is provided below, All the media compositions given below are for 100 ml volume. Compositions changes depending upon the quantity required,

0) Plggamtlpnofll brplh..(pH^.6J}

-Composition ofT3 medium

All the media components were weighed and taken in a glass bottle and dissolved in distilled water. The container with the medium was autoclaved at 121^VC for 15 min.

(«) Prgpgratic»^^

Composition of T3 medium

Ail the media components were weighed and taken in a glass bottle and dissolved in distilled water. The glass bottle with the medium was autoclaved at 12 PC for 15 min. Alter autoclaving, the medium was poured in to sterile petri plates $3

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Composition of LB medium

Ali the media components were weighed and taken in a glass bottle and dissolved in distilled water. The container with the medium was autoclaved at 12. PC for 15 min.

11^v) fooparatfonof |,B .a^phucs .(m_;LnX0) Composition of LB medium

All the media components were weighed and taken in a glass bottle and dissolved in distilled water. The glass bottle with the medium was autoclaved at. 121 °C for 15 min. After autoclaving, the medium was poured in to sterile petri plates,

IV)

Composition of PDB medium

S. No.	Component	Quantity
Ifo a	Potato Powder	2,0 % (w'v)
2 :	Dextrose	2.0% (w'v)
3 ;	Speetlnomycin (Optional)	100 pg/ml

MsM

Potato powder (2.0 g) was weighed and taken in a 250 mi glass bottle containing 50 mi of distilled water and boiled for 5 min. The boiled potato water was filtered using muslin cloth. The- .filtrate was collected in a fresh 250 mi glass bottle and 2.0 g of dextrose was added to it. Alter making the total volume to 100 mi, the bottle with the medium was autoclaved at I2DC for 1S mm.

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M.M.PDA agarjxUtgi(Rll^6O)

Composition of PDA medium

S. No.	Component	Quantity
1	Potato Powder	2.0 % (w/v)
2	Dextrose	2.0 % (w/v)
3	Agar agar	2,0 % (w/v)
4	Speettnomyem ^Optional)	100 gg/ml

Method

Potato powder (2,0 g) was weighed and taken in a 250 ml glass bottle containing 50 mi of distilled water and boiled for'5 min. The boiled potato water was filtered using muslin cloth. The filtrate was collected in a fresh 250 ml glass bottle and 2.0 g of dextrose and 2.0 g of agar were added to it. After making the total volume to 100 nth the bottle with the medium was autoclaved at 121 ^ftC for 15 min. The molten PDA was poured into the sterile petri plates.

Composition of OF SM medium

S. No.	Component	Quantity
i	Casein Peptone (Trvptone) 0,2%	0,2 % (w/v)
2	NaCl	0.5 % (w/v)
3	KTiPo,! ......	0,03 % (w/v)
4	Agar agar	2,0% (w/v)
5	Bromothymol Blue	0,004% (w/v)

Method

All the media components were weighed and taken in a glass bottle and dissolved in distilled water. The glass bottle with the medium was autoclaved at 121°C for 15 min. After autoclaving, the medium was poured in to sterile culture tubes.

(VI) IheoMatiotlof SlM..(Sufohlde Indole MptfeXjngdipm (pR-.JJl

Composition of SIM medium

S, No.	Component	Quantity
1	Peptone	3.0 % (w/v)
2	Beef Extract	0,3 % (w/v)
3	Ferrous Ammomum Sulphate	0.02 % (w/v)
4	Sodium thiosu 1 hphatc	0,0025 % (w/v)
7	Agar agar	2,0 % (w/v)

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Method

Ah the media components were weighed end taken in a glass bottle and dissolved in distilled water. The glass bottle with the medium was autoclaved at 12 PC for 15 rain. After autoclaving, the medium of molten stage was poured in to sterile culture tubes.

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Annexure II

Preparation phosphate buffer

S.No.	Component	Quantity	Weight for 100 ml
4	NaHiPCM.HiO	1 M	5.38 g
5	Na2HPO4	1 M	8.66 g
	pH	7.0

Method

Both the phosphate salts were taken in a glass beaker, 50 ml distilled water was added to salts and stirred on a magnetic stirrer using a magnetic bar. After ensuring that the phosphate salts are completely dissolved, the solution was made up to 100 ml with distilled water.

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Annexure III

Carriers and other agents used in the experiment

S.No.	Agent Name	Composition	Used as/for	Make
1	CMC	Carboxy Methyl Cellulose	Binding agent	Himedia
2	Carbendazim	Carbendazim WP50	fungicide	Bavistin
3	Soilrite	Perlite+ Peat Moss+ Vermiculite	Fungus multiplication	KEL (Keltech Energies Ltd)

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S,No.	Short Form	Full Form
1	ATCC	American Type Culture Collection
2	CMC	Carboxy Methyl Cellulose
3	CFU	Colony Forming Units
4	L	Liter
5	LB	Lnria-Bertnni
<5	M / : c-	Micro Liter
7	MTCC	Microbial Type Culture Collection
8	Mi	Midi Liter
9	MIC	Minimal inhibitory Concentration
10	Min	Minutes
ii	M	Molar
n	NPCCi	National Fungal Culture Collection of India
13	OFBM	Oxidation Fermentation Basal Medium
14	PDA	Potato Dextrose Agar
15	PDB	Potato Dextrose Broth
16	RPM	Revolutions Per Minute
17	Ssp	Sub-species
'18	SIM	Sulphide Indole Motility
19	v/v .	Yoiume by volume
20	w/v	Weight bv volume
21	w/w	Weight by weight
22	WP	Wettabie Powder

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Throughout this specification and the claims which follow, unless the context requires otherwise, the word comprise, and variations such as comprises or comprising, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

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Claims (14)

1. The claims defining the invention are as follows:

   1. A novel bacterium belonging to a Bacillus species exhibiting antimicrobial and/or antifungal activity and plant growth promoting activity, wherein the novel bacterium is isolated Bacillus subtilis ssp. shriramensis having the accession number MTCC-5674.
2. 2. An extract of the novel bacterium belonging to a Bacillus species as claimed in claim 1, exhibiting antimicrobial and/or antifungal activity.
3. 3. A pure culture of the novel bacterium as claimed in claim 1.
4. 4. A process for producing the extract as claimed in claim 2, wherein the process comprises:

   a. growing the Bacillus subtilis ssp. shriramensis having the accession number MTCC-5674 in a T3 medium having pH 6.8 in a shaking incubator at 30°C for 60 h; and

   b. recovering the extract having antimicrobial and/or antifungal activity.
5. 5. The process as claimed in claim 4, wherein the Bacillus subtilis ssp. shriramensis is grown under aerobic conditions.
6. 6. The process as claimed in claim 4 or 5, wherein the process further comprises concentrating the extract using conventional methods.
7. 7. A composition comprising the novel bacterium as defined in claim 1, wherein the composition has antimicrobial and/or antifungal activity and plant growth promoting activity at a concentration between about 5xl0⁵ cfu/ml to about 5xl0⁷ cfu/ml of the bacterium.
8. 8. A composition comprising the extract as claimed in claim 2, wherein the composition has antimicrobial and/or antifungal activity at a concentration of between about 4 pg/μΐ to about 20 pg/μΐ of the extract.

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9. 9. A composition comprising the novel bacterium as claimed in claim 1 and the extract as defined in claim 2, wherein the composition has antimicrobial and/or antifungal activity and plant growth promoting activity.
10. 10. The composition as claimed in any one of claims 7 to 9, wherein the composition further comprises one or more antimicrobial and/or antifungal agents and/or plant growth promoting agents.
11. 11. The composition as claimed in any one of claims 7 to 10, wherein the composition further comprises an agriculturally acceptable carrier.
12. 12. A method for inhibiting growth of pathogenic fungi and/or bacteria, wherein said method comprises contacting the pathogenic fungi and/or bacteria with an effective amount of between about 5xl0⁵ cfu/ml to about 5xl0⁷ cfu/ml of the novel bacterium as claimed in claim 1 or a composition as defined in any one of claims 7 to 11.
13. 13. The novel bacterium as claimed in claim 1 or the extract as claimed in claim 2 when used for the preparation of a composition for inhibiting the growth of pathogenic fungi and/or bacteria.

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    Control with water No clearance zone

    Hydrolysis of starch by extract

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